Auxotrophy, the inability of bacteria to synthesize one or multiple essential metabolites, is thought to be common among bacteria. However, studies often rely on either bioinformatic genome-based prediction of auxotrophies or on experiments with low strain numbers. Here, we combine experimental and bioinformatic approaches to assess amino acid auxotrophies among 315 co-isolated natural Pseudomonas strains from pond and soil habitats. Experiments revealed that Pseudomonas isolates are predominantly prototrophs. We identified one histidine auxotroph, one auxotroph with complex intertwined dependencies and four 'fragile' phenotypes showing delayed growth without supplemented amino acids. Applying three bioinformatic pipelines largely confirmed experimental data but yielded specific biases in auxotrophy over- or underestimation. Moreover, none of the pipelines could resolve the genetic basis of the slow-growing phenotypes or of more complex dependencies. Our analysis further revealed the existence of multiple alternative biosynthesis pathways for methionine, proline and phenylalanine, with significant pathway enrichments being linked to phylogeny and habitat. We conclude that combining experiments with bioinformatics is a powerful approach to assess the metabolic potential of environmental bacteria. Moreover, taxa like Pseudomonas can be predominantly prototrophic possibly owing to their generalist lifestyle, thus calling for nuanced ecological concepts predicting auxotrophy levels based on lifestyle and habitat.

YABBY transcription factors are an essential class of plant-specific macromolecules that regulate cellular processes, response to stress, secondary metabolite biosynthesis, and developmental regulation. Despite their significance in plant macromolecular networks, their roles in woody perennials remain underexplored. In this study, we identified and classified 13 YABBY proteins from Populus trichocarpa and 22 YABBY proteins from P. tomentosa into five distinct subfamilies. Subsequently, we constructed a phylogenetic tree based on the YABBY proteins of five plant species, and we analyzed the conserved structural domains, motifs, promoter regions, and collinearity of the poplar YABBYs. Expression patterns of the poplar YABBYs were characterized in vegetative organs and male/female flower buds. The results indicated that PtoYABBY6 is predominantly expressed in reproductive organs, with higher expression levels observed in female flower buds compared to male flower buds. Functional analysis of PtoYABBY6 demonstrated that its overexpression in Arabidopsis thaliana led to partial alterations in the adaxial-abaxial axis polarity of leaves and delayed flowering. Additionally, the expression levels of FLOWERING LOCUS T (FT), a gene associated with flowering time, as well as FILAMENTOUS FLOWER (FIL), KANADI (KAN), REVOLUTA (REV), and PHAVOLUTA (PHV; genes related to leaf adaxial-abaxial polarity) were altered in Arabidopsis. Furthermore, transient overexpression of PtoYABBY6 in poplar leaves induced subtle changes in adaxial epidermal cell morphology and increased the palisade-to-spongy tissue ratio, providing partial evidence suggesting a potential role in leaf development within a poplar cellular context. These findings provide a foundation for future functional studies and contribute to a better understanding of the molecular mechanisms underlying YABBY protein functions in poplar.

The zinc finger transcription factor PLAG1 has been implicated in multiple diverse cellular processes, yet its role in the onset of vertebrate neurodevelopment and behaviour remains largely unexplored. Notably, dysregulation of PLAG1 has been associated with Silver-Russell syndrome (SRS) in humans, a growth disorder often presenting with neurodevelopmental delay and craniofacial abnormalities. Here, we investigate the function of plag1 in zebrafish embryogenesis, focusing on behavioural phenotypes, neural development and craniofacial morphogenesis, as a potential novel model of SRS. Using antisense morpholino-mediated knockdown, we assessed the impact of plag1 loss on brain architecture, motor neuron development, craniofacial patterning and stimulus-evoked behavioural responses. While plag1-deficient embryos displayed pronounced locomotor defects when housed in groups, these phenotypes were not recapitulated under isolated, individual tracking conditions, suggesting a role for plag1 in modulating specific behavioural outputs. Morphological analyses revealed no overt disruptions to gross brain structure or primary motor neuron patterning. However, quantitation of craniofacial skeleton formation identified subtle abnormalities in jaw and cranial cartilage development, consistent with features observed in SRS. Furthermore, plag1 morphants exhibited significantly altered responses to sensory stimuli, implicating plag1 in the regulation of sensorimotor integration during early development. Collectively, these findings uncover a novel role for plag1 in early neurobehavioural modulation and craniofacial patterning, phenocopying multiple features of SRS, and highlight the importance of environmental and social context in the phenotypic interpretation of neurodevelopmental gene function.

Eosinophilic gastrointestinal diseases (EGIDs) are chronic, TH2-mediated conditions. Eosinophilic esophagitis (EoE) is the most common type, while non-EoE EGIDs affect other gut segments. This retrospective study of 111 patients (50 EoE and 61 non-EoE) from 2011 to 2022 compared the clinical aspects of these 2 types. Dysphagia and food impaction dominated in EoE, while abdominal pain, nausea, and diarrhea were more common in non-EoE EGIDs. Atopic comorbidities were frequent. Diagnostic delays>1 year were more common in EoE (72% vs 47.5%). The overall clinical response rate was 88.29%, with most patients using food avoidance (90.99%) and proton pump inhibitors (94.59%). Clinical relapses occurred in 38.88% over the mean follow-up of 4.62 years and were unaffected by therapy type. Failure to thrive was seen in 26.7%, with no significant intergroup difference. This study highlights a prolonged diagnostic delay in EoE vs non-EoE EGIDs. Both groups showed similar, favorable response rates, underscoring a need for greater awareness. Prospective studies with standardized measures are required to optimize management.

Diabetic foot ulcer (DFU) is a severe complication of diabetes, characterized by chronic, difficult‑to‑heal foot wounds. It has been hypothesized that miR‑222‑3p may influence wound healing via the regulation of angiogenesis. To elucidate the underlying mechanism, experimental investigations were conducted in vitro using human dermal microvascular endothelial cells (HMEC‑1) and in vivo in a mouse model of streptozotocin‑induced diabetes. Methylglyoxal (MGO), a glycolytic intermediate, is known to contribute to diabetic vascular complications. The results demonstrated that MGO‑treated HMEC‑1 cells exhibited an increased expression of miR‑222‑3p and impaired microvascular endothelial cell function. The overexpression of miR‑222‑3p in MGO‑treated HMEC‑1 cells further suppressed cell proliferation, migration and tube formation, while it enhanced apoptosis. Moreover, the upregulation of miR‑222‑3p reduced the expression of apelin (APLN), and dual‑luciferase reporter assays confirmed APLN as a direct downstream target of miR‑222‑3p. The silencing of APLN markedly impaired HMEC‑1 cell function. Notably, both the exogenous supplementation with Apelin‑13 and the overexpression of APLN significantly reversed endothelial dysfunction caused by the upregulation of miR‑222‑3p. In vivo, in both normal and diabetic mice, the elevated expression of miR‑222‑3p in skin wounds reduced APLN levels in the peri‑wound tissue and impaired wound healing. However, the overexpression of APLN or the inhibition of miR‑222‑3p with an antagomiR reversed this impairment. Collectively, these results suggest that miR‑222‑3p impairs microvascular endothelial cell function by targeting APLN, thereby contributing to delayed wound healing in diabetic skin.

Silent corticotroph adenomas (SCAs) are a biologically distinct subset of nonfunctioning pituitary adenomas (NFPAs) characterized by corticotroph lineage without clinical or biochemical evidence of hypercortisolism. Although SCAs exhibit more aggressive growth behavior than other NFPAs, delayed transformation into overt adrenocorticotropic hormone (ACTH)-dependent hypercortisolism is rare. We describe a woman in her early 30s who presented with headaches and visual field deficits and was found to have a pituitary macroadenoma. Initial endocrine evaluation revealed mild hyperprolactinemia attributed to stalk effect, without evidence of hormonal hypersecretion. She underwent transsphenoidal surgery, and pathology was interpreted as an NFPA based on negative immunostaining. Transcription factor analysis was not performed. Postoperative imaging demonstrated a stable 5-mm residual lesion. More than 2 decades after surgery, progressive ACTH elevation emerged despite continued radiographic stability and preceded the development of clinical hypercortisolism by several years. Biochemical testing confirmed ACTH-dependent Cushing disease. Repeat surgery was performed and surgical pathology showed a T-box transcription factor-positive corticotroph adenoma with sparsely granulated ACTH expression. This case highlights the potential for very late functional transformation of SCAs and underscores the importance of long-term clinical and biochemical surveillance even in asymptomatic patients with stable residual disease on imaging.

The clinical management of Candida albicans (C. albicans) infections presents considerable problems, intensified by the few therapeutic choices available for this disease. The increasing resistance to fluconazole (FLC) following its widespread clinical use underscores the need for novel therapeutic strategies. Drug repurposing represents a promising approach to expand the antifungal armamentarium, and bortezomib (BTZ), a proteasome inhibitor originally approved for multiple myeloma, has emerged as a candidate for this purpose. This work systematically assessed the synergistic effects of fluconazole (FLC) with bortezomib (BTZ) on the susceptibility of C. albicans, focusing specifically on the planktonic and biofilm development phases. The results exhibited a synergistic antifungal effect of BTZ and FLC against FLC-resistant C. albicans in planktonic cells. The minimal inhibitory concentration (MIC) of BTZ decreased from 64-128 µg/ml to 8 µg/ml, whereas the MIC of FLC reduced from >128 µg/ml to 0.25 µg/ml, resulting in a fractional inhibitory concentration index (FICI) of less than 0.5. For FLC-susceptible C. albicans strains, the medication combination exhibited an indifferent interaction, with FICI above 0.5. The combination exhibited synergistic effects against C. albicans during the early stages of biofilm formation (4 and 8 h). The sessile minimum inhibitory concentration (sMIC) of FLC significantly lowered from ≥128 µg/mL to 0.25 µg/mL, whereas the sMIC of BTZ diminished from ≥128 µg/mL to 8-16 µg/mL, with a FICI of less than 0.5, signifying significant synergy. Time-kill assays revealed a delayed but sustained inhibitory effect against planktonic cells (24-48 h), while CFU enumeration confirmed a concentration-dependent reduction in biofilm viability, with the higher concentration (0.25 + 16) maintaining efficacy through 24 h. In vivo, using the Galleria mellonella (G. mellonella) infection model, the FLC+BTZ combination significantly improved larval survival, reduced fungal burden, and alleviated tissue damage compared with FLC monotherapy. Mechanistic studies demonstrated that this synergistic effect was mediated by multiple mechanisms, including the suppression of hyphal growth, inhibition of efflux pump activity, and intracellular formation of reactive oxygen species (ROS). These findings highlight the potential of drug repurposing in combating antifungal resistance and provide mechanistic insights into overcoming C. albicans drug resistance.

Regenerative capacity varies widely across animals, yet aging is often accompanied by declining tissue homeostasis and regenerative potential. In many species, regeneration of complex structures relies on epimorphic programs that form a blastema, coordinating cell proliferation and pattern reorganization after injury. Although aging-associated regeneration defects have been documented in several bilaterian models, how aging shapes regeneration in early-branching metazoans with robust regenerative abilities remains unclear. Here, we investigate aging and regeneration in the medusa stage of Cladonema pacificum, a cnidarian that retains high regenerative capacity within a finite lifespan. We show that aging in Cladonema medusae is accompanied by progressive morphological and functional deterioration, including shrinkage of the umbrella and manubrium, tentacle shortening, and reduced reproductive output, along with increased DNA damage. At the cellular level, aging is associated with depletion of differentiated cell populations, including nematocytes and neurons, as well as reductions in stem cell-associated populations and proliferative activity in the tentacle bulb. Consistent with these changes, tentacle regeneration is markedly impaired in aged medusae and is characterized by delayed wound closure, defective blastema formation and incomplete functional recovery. Together, our findings indicate that aging disrupts both tissue homeostasis and blastema-mediated regeneration in Cladonema medusae, establishing a tractable model for studying aging-regeneration interactions and supporting the view that aging imposes broadly shared constraints on regenerative systems across metazoans.

BackgroundWomen living in rural and remote communities face longstanding barriers to accessing prenatal diagnostic care, including long travel distances, limited appointment availability, and delayed diagnostic evaluation. Telerobotic ultrasound may improve prenatal care by expanding access through imaging in local communities. However, little is known about women's lived experiences with this technology and providers' perspectives on cultural safety within prenatal care.ObjectivesTo explore women's and providers' experiences and perceptions of telerobotic ultrasound in prenatal care.DesignQualitatively driven mixed-methods study.MethodsPostpartum women with experience using or declining telerobotic ultrasound, along with providers involved in service delivery in Gravelbourg and Saskatoon, participated in semi-structured remote interviews and were offered nine-point Likert-scale surveys. Discussion topics included decision-making, cultural and linguistic considerations, and integration of telerobotic imaging into rural care. Interviews were transcribed and analyzed using inductive coding, constant comparison, and framework-informed interpretation in NVivo 15.ResultsA total of 40 participants took part in the study, including 19 patient participants and 21 provider participants, all of whom completed interviews. Survey findings, based on 7 sufficiently complete patient surveys and 10 provider surveys returned within the data-collection period, supplemented the qualitative findings. Participants reported improved access to prenatal care, particularly through reduced travel and local service availability. However, limited awareness, inconsistent communication, and gaps in knowledge were identified as key barriers to uptake. Participants also highlighted challenges related to cultural and linguistic accommodation and reliance on technological infrastructure. Cultural and linguistic aspects were often characterized by uncertainty, with many patients reporting neutral views on cultural sensitivity and language accessibility. Patients expressed mixed levels of trust and information sufficiency, while providers reported confidence in image quality and workflow integration but identified gaps in language accessibility and patient preparedness.ConclusionTelerobotic ultrasound in prenatal diagnostic care was perceived as acceptable and potentially beneficial for improving access, but attention to cultural safety, communication, workflow alignment, and reliable technical support will be important when expanding service delivery.

Ageing is a primary risk factor for neurodegenerative disorders, including Parkinson's disease (PD). As individuals age, their cells become less efficient in maintaining protein homeostasis, leading to an increased likelihood of protein misfolding and aggregation. A hallmark of PD and other synucleinopathies is the accumulation of alpha-synuclein protein aggregates in affected neurons, a process that is exacerbated by ageing. While cellular mechanisms that regulate protein aggregation have been a primary focus of research, recent studies suggest that other, systemic age-related mechanisms may contribute to alpha-synuclein toxicity. Understanding these alternative pathways is crucial for the development of effective therapeutic strategies to combat neurodegenerative diseases, such as PD. In this review, we synthesize current insights into the biological mechanisms underlying alpha-synuclein toxicity at the organismal level. We highlight key open questions and discuss how these findings may inform the development of targeted interventions to prevent or delay age-related synucleinopathies. This article is part of the Theo Murphy meeting issue 'ProteostaSys: a systems view of proteostasis'.

In vivo genome editing utilizing Cas9/sgRNA ribonucleoproteins (RNPs) holds substantial therapeutic promise, yet rapid bloodstream clearance and degradation of RNPs have hindered its accumulation within the target site and effective gene editing. Herein, we report a supramolecular ternary complex platform where RNPs are co-assembled with tannic acid (TA) and phenylboronic acid (PBA)-conjugated polymers through sequential self-assembly. This ∼30 nm core-shell structure protects RNPs from enzymatic degradation and dissociates selectively at endosomal pH. Upon intravenous administration in subcutaneous tumor-bearing mice, the ternary complex exhibits prolonged blood circulation and preferential tumor accumulation, achieving 37.2% gene editing at tumor sites compared with only 1.5% for free RNPs. The platform successfully disrupts PLK1 in subcutaneous tumors, accompanied by significant tumor growth suppression. This is further applied to a KRASG13D-mutant tumor model, in which treatment delayed tumor growth. By integrating sequential supramolecular self-assembly with stimuli-responsive cargo release, this strategy presents a promising approach for systemic RNP delivery and tumor genome editing.

Understanding retinal development is important for better explaining retinal function and disease and as a model for generating retinal organoids and retinal regeneration strategies. Although development of mouse retina is studied in great detail, timing of retinal cell type development in humans is still poorly understood. This study aims to compare developmental steps in the human midgestational and mouse early postnatal retinas and to establish timeline correspondents and their anatomical landmarks. Immunofluorescence analysis of retinal cryosections was performed on human retinas collected from fetuses of 15 to 27 gestational weeks (GW15-GW27) and mouse retina from pups ranging from 0 to 14 days (P0-P17). Markers for retina vascularization, cell type development, and layer lamination were determined and compared across species and developmental ages. Vascularization emerges and progresses over the investigated developmental timespan in both species. In parallel, lamination of nuclear layers progresses from two (ganglion cell layer = GCL vs. neuroblast layer = NBL) layers to the mature pattern of three layers (GCL-inner nuclear layer = INL-outer nuclear layer = ONL). Molecular markers of retinal cell types and markers of inner plexiform layer (IPL) lamination are appearing at the onset of the studied time points and progressively settle into the adult pattern of localization. A strong central-to-peripheral delay is observed in human retinas at GW18 and GW20. It therefore appears that human midgestation retina development is mirrored in the first 2 weeks of mouse postnatal development. The present work closes a gap in the literature concerning the correlation between human and mouse retinal maturation timelines. The stages identified and their anatomical landmarks may provide as a basis for future comparative studies.